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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-12-01 to 2015-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD Guideline 201 (March 2006)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 125, 250, 500, 1000 and 2000 mg/L test substance
- Sampling method: 20 ml taken daily from test vessels without algae directly from the test vessel and after 72 h with algae, stabilized wit 200 µl of nitric acid
- Sample storage conditions before analysis: 5°C ± 3°C (additional retain samples at ≤ -18°C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct addition to algal medium for preparation of stock solution (2000 mg/L), stirred for 24 h, subsequently pH was adjusted to 6.5 using 5N and 0.5N NaOH, The occurring precipitations were allowed to settle and the clear supernatant was decanted and filtered through a 0.45 µm membrane filter. The filtrated test solution was diluted with algal medium to obtain the nominal loading rates of 125 to 1000 mg/L.
- Eluate: Algal growth medium
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Highly varying Ta5+ concentrations in solution depending on pH according to ananalytical analysis.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Göttingen, subcultured at Hydrotox GmbH since November 2013
- Age of inoculum (at test initiation): 3 d (57.99 x 10^5 algal cells/ml after 3 d)
- Method of cultivation: Suspension culture in Holm-Hansen medium under axenic conditions

ACCLIMATION
- Three days before starting the test, 2.778 mL of the algae stock suspension was diluted into 50 mL algal growth medium (according to OECD 201) to obtain a starting concentration of 1 x 10^5 algal cells per mL. They were incubated at 22.9 – 23.3°C and 118 μE/m² s +/- 6,0% (PAR). After three days, the cell concentration was determined using the Coulter Counter Z2. The cell concentration was 57.99 x 10^5 algal cells/mL. To obtain a nominal inoculum concentration of 0.7 x 10^5 algal cells/mL 5.43 mL pre-culture was diluted into 450 mL algal growth medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.7 to 23.2°C
pH:
See Table 2.
Salinity:
Due to the required neutralization of the medium containing tantalum pentachloride, the salinity increased mainly because of the formation of NaCl to a maximum concentration of 2.92 g/L at the highest nominal test concentration of 2000 mg/L. The salinity is accordingly lower in samples of lower nominal test item concentrations.
Nominal and measured concentrations:
See Table 3.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml wide-necked Erlenmeyer flask
- Type: closed
- Fill volume: 50 ml
- Aeration: no
- Initial cells density: 7 x 10^3 algae ml-1
- Control end cells density: 1.886 x 10^5 ml-1
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Medium used: Holm-Hansen medium

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: 117.6 µE/m2 s +/- 4.6% (PAR)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Measurement of chlorophyll fluorescence to be converted into cell counts per ml by means of a correlation factor
- Chlorophyll measurement: Chlorophyll fluorescence

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 100 mg/L and 1000 mg/L test substance inhibited growth rates by 25% and 80 %
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 2 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
464 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 218 – 712 mg/L)
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
122 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 19.0 – 247 mg/L)
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
504 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (95%-CI: 266 – 960 mg/L)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- After pH adjustment the measured Ta5+ concentrations were low and showed high variability. Therefore, no concentration-response relationship could be established. Concentrations of the test sample varied due to precipitation, agglomeration and adsorption reactions. In addition, slight changes in pH had profound impact on the test concentrations. Observed effects may therefore predominantly refer to elevated NaCl concentrations originated from neutralization, than released Ta5+ ions. Hypothetical Ta5+ related effects cannot be distinguished from NaCl induced inhibition in the conducted test. Therefore, only nominal effect levels, respectively nominal loading rates, were calculated.

Results with reference substance (positive control):
- ErC50: 0,74 mg/l (0,73 – 0,76 mg/L 95%-CI)
- EyC50: (Yield) 0,36 mg/l (0,35 – 0,36 mg/l 95%-CI).

Table 2: Mean growth rates, inhibition of growth rates and yield, as well as measured pH

Sample code

Control

A

B

C

D

E

Nominal loading rate [mg/L]

0

125

250

500

1000

2000

No. of replicates

6

3

3

3

3

3

Mean growth rate

1.10

0.99

0.95

0.81

0.88

0.67

Mean yield

1.82

1.34

1.15

0.72

0.92

0.45

Mean inhibition of growth rate [%]

-

10.0

13.6

26.5

19.6

39.4

Mean inhibition of yield [%]

-

26.4

36.5

60.3

49.4

75.2

pH

0 h

6.4

6.6

6.6

6.7

6.7

6.7

72 h

7.1

7.0

7.1

7.1

7.1

7.1

Table 3: Nominal and measured concentrations of the test substance tantalum pentachloride.

Sample Code

NC

A

B

C

D

E

Nominal loading rate [mg/L]

0

125

250

500

1000

2000

Measured concentration [µg/L]

0 h

< LOQ

< LOQ

< LOQ

2.38

3.81

11.7

24 h

< LOQ

< LOQ

< LOQ

< LOQ

0.96

< LOQ

48 h

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

72 h

< LOQ

< LOQ

2.70

< LOQ

2.10

35.0

72 h*

< LOQ

8.35

5.87

2.97

9.76

14.4

LOQ (Limit of quantification) = 0.5 μg/L

*from test vessels with algae

72 h samples were analysed after chemical digestion

Validity criteria fulfilled:
yes
Conclusions:
For the conducted static algae growth inhibition test with Pseudokirchneriella subcapitata, applying nominal concentrations of 0 (control), 125, 250, 500, 1000 and 2000 mg/L tantalum pentachloride, results based on growth rate after 72 h were as follows: EL50: > 2000 mg/L; EL20: 464 mg/L (95 % C.l. 218 – 713 mg/L); EL10: 122 mg/L (95 % C.l. 19.0 – 247 mg/L); LOELR: 500 mg/L; NOELR: 250 mg/L. The values indicate effects at nominal loading rates. Due to methodological constraints, it was not possible to determine effect levels based on measured test item concentrations.
Executive summary:

In a 72 -h growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to tantalum pentachloride at nominal concentrations of 0 (control), 125, 250, 500, 1000 and 2000 mg/L under static conditions in accordance with the OECD Guideline 201 (March 2006) and under GLP. The % growth inhibition in the treated algal culture as compared to the control ranged from 10.0 % at a nominal concentration of 125 mg/L to 39.4 % at a nominal concentration of 2000 mg/L. Specific results based on growth rate after 72 h were as follows: EL50: > 2000 mg/L; EL20: 464 mg/L (95 % C.l. 218 – 713 mg/L); EL10: 122 mg/L (95 % C.l. 19.0 – 247 mg/L); LOELR: 500 mg/L; NOELR: 250 mg/L. The values indicate effects at nominal loading rates. Due to methodological constraints, it was not possible to determine effect levels based on measured test item concentrations. After pH adjustment the measured Ta5+ concentrations were low and showed high variability. Measured concentrations were more than a factor of 1000 lower than in the respective nominal loading rates after pH adjustment. Measured concentrations were mostly below the limit of quantification (LOQ) in samples that were not chemically digested. Only at 0 h, measured concentrations were with 2.38 to 11.7 µg/L in the three highest loading rates above the LOQ. Samples taken at the end of the test (72 h) were analysed after being chemically digested to analyse the total (soluble plus insoluble) test item concentration. Here, values ranged from 2.97 to 14.4 µg/L, but no relation between measured concentration and the nominal loading rate could be observed. Concentrations of the test sample varied due to precipitation, agglomeration and adsorption reactions. Therefore, no concentration-response relationship can be delineated.

In addition, slight changes in pH had profound impact on the test concentrations. Observed growth inhibition effects may therefore rather predominantly refer to elevated NaCl concentrations, originated from neutralization, than released Ta5+ ions. Reported effects of sodium chloride concerning the freshwater alga P. subcapitata vary but inhibitory concentrations of 2.5 g/L, respectively 0.87 g/L have been determined (Geis et al., 2000 and Santos et al., 2007, in Heisterkamp, 2015). Due to a concentration of approximately 2.92 g/L NaCl, present at the highest loading rate of 2000 mg/L test substance, and the sensitivity of P. subcapitata to salinity, any hypothetical Ta5+ related effects cannot be distinguished from NaCl induced inhibition in the conducted test. This study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata (Strain No. 61.81 SAG)

Test Type: Static

 

Specific Growth Rate
72 h nominal loading rate

Yield

72 h nominal loading rate

ELR50

> 2000 mg/L

504 mg/L

(95 % C.I. 266 – 960 mg/L)

ELR20

464 mg/L

(95 % C.I. 218 – 713 mg/L)

n.d.

ELR10

122 mg/L

(95 % C.I. 19.0 – 247 mg/L)

n.d.

Slope of the curve

0.758

0.924

P(Chi²)

1.000

1.000

0.604

0.526

LOELR

500 mg/L

n.d.

NOELR

250 mg/L

n.d.

n.d.: not determined by statistical analysis

Endpoint(s) Effected:  Algal growth and biomass.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For details and justification of read-across please refer to the attached report in section 13 of IUCLID.
Reason / purpose:
read-across source
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 2 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
464 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 218 – 712 mg/L)
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
122 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 19.0 – 247 mg/L)
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
504 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (95%-CI: 266 – 960 mg/L)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- After pH adjustment the measured Ta5+ concentrations were low and showed high variability. Therefore, no concentration-response relationship could be established. Concentrations of the test sample varied due to precipitation, agglomeration and adsorption reactions. In addition, slight changes in pH had profound impact on the test concentrations. Observed effects may therefore predominantly refer to elevated NaCl concentrations originated from neutralization, than released Ta5+ ions. Hypothetical Ta5+ related effects cannot be distinguished from NaCl induced inhibition in the conducted test. Therefore, only nominal effect levels, respectively nominal loading rates, were calculated.

Results with reference substance (positive control):
- ErC50: 0,74 mg/l (0,73 – 0,76 mg/L 95%-CI)
- EyC50: (Yield) 0,36 mg/l (0,35 – 0,36 mg/l 95%-CI).

Table 2: Mean growth rates, inhibition of growth rates and yield, as well as measured pH

Sample code

Control

A

B

C

D

E

Nominal loading rate [mg/L]

0

125

250

500

1000

2000

No. of replicates

6

3

3

3

3

3

Mean growth rate

1.10

0.99

0.95

0.81

0.88

0.67

Mean yield

1.82

1.34

1.15

0.72

0.92

0.45

Mean inhibition of growth rate [%]

-

10.0

13.6

26.5

19.6

39.4

Mean inhibition of yield [%]

-

26.4

36.5

60.3

49.4

75.2

pH

0 h

6.4

6.6

6.6

6.7

6.7

6.7

72 h

7.1

7.0

7.1

7.1

7.1

7.1

Table 3: Nominal and measured concentrations of the test substance tantalum pentachloride.

Sample Code

NC

A

B

C

D

E

Nominal loading rate [mg/L]

0

125

250

500

1000

2000

Measured concentration [µg/L]

0 h

< LOQ

< LOQ

< LOQ

2.38

3.81

11.7

24 h

< LOQ

< LOQ

< LOQ

< LOQ

0.96

< LOQ

48 h

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

72 h

< LOQ

< LOQ

2.70

< LOQ

2.10

35.0

72 h*

< LOQ

8.35

5.87

2.97

9.76

14.4

LOQ (Limit of quantification) = 0.5 μg/L

*from test vessels with algae

72 h samples were analysed after chemical digestion

Validity criteria fulfilled:
yes
Conclusions:
For the conducted static algae growth inhibition test with Pseudokirchneriella subcapitata, applying nominal concentrations of 0 (control), 125, 250, 500, 1000 and 2000 mg/L tantalum pentachloride, results based on growth rate after 72 h were as follows: EL50: > 2000 mg/L; EL20: 464 mg/L (95 % C.l. 218 – 713 mg/L); EL10: 122 mg/L (95 % C.l. 19.0 – 247 mg/L); LOELR: 500 mg/L; NOELR: 250 mg/L. The values indicate effects at nominal loading rates. Due to methodological constraints, it was not possible to determine effect levels based on measured test item concentrations.
Executive summary:

In a 72 -h growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to tantalum pentachloride at nominal concentrations of 0 (control), 125, 250, 500, 1000 and 2000 mg/L under static conditions in accordance with the OECD Guideline 201 (March 2006) and under GLP. The % growth inhibition in the treated algal culture as compared to the control ranged from 10.0 % at a nominal concentration of 125 mg/L to 39.4 % at a nominal concentration of 2000 mg/L. Specific results based on growth rate after 72 h were as follows: EL50: > 2000 mg/L; EL20: 464 mg/L (95 % C.l. 218 – 713 mg/L); EL10: 122 mg/L (95 % C.l. 19.0 – 247 mg/L); LOELR: 500 mg/L; NOELR: 250 mg/L. The values indicate effects at nominal loading rates. Due to methodological constraints, it was not possible to determine effect levels based on measured test item concentrations. After pH adjustment the measured Ta5+ concentrations were low and showed high variability. Measured concentrations were more than a factor of 1000 lower than in the respective nominal loading rates after pH adjustment. Measured concentrations were mostly below the limit of quantification (LOQ) in samples that were not chemically digested. Only at 0 h, measured concentrations were with 2.38 to 11.7 µg/L in the three highest loading rates above the LOQ. Samples taken at the end of the test (72 h) were analysed after being chemically digested to analyse the total (soluble plus insoluble) test item concentration. Here, values ranged from 2.97 to 14.4 µg/L, but no relation between measured concentration and the nominal loading rate could be observed. Concentrations of the test sample varied due to precipitation, agglomeration and adsorption reactions. Therefore, no concentration-response relationship can be delineated.

In addition, slight changes in pH had profound impact on the test concentrations. Observed growth inhibition effects may therefore rather predominantly refer to elevated NaCl concentrations, originated from neutralization, than released Ta5+ ions. Reported effects of sodium chloride concerning the freshwater alga P. subcapitata vary but inhibitory concentrations of 2.5 g/L, respectively 0.87 g/L have been determined (Geis et al., 2000 and Santos et al., 2007, in Heisterkamp, 2015). Due to a concentration of approximately 2.92 g/L NaCl, present at the highest loading rate of 2000 mg/L test substance, and the sensitivity of P. subcapitata to salinity, any hypothetical Ta5+ related effects cannot be distinguished from NaCl induced inhibition in the conducted test. This study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata (Strain No. 61.81 SAG)

Test Type: Static

 

Specific Growth Rate
72 h nominal loading rate

Yield

72 h nominal loading rate

ELR50

> 2000 mg/L

504 mg/L

(95 % C.I. 266 – 960 mg/L)

ELR20

464 mg/L

(95 % C.I. 218 – 713 mg/L)

n.d.

ELR10

122 mg/L

(95 % C.I. 19.0 – 247 mg/L)

n.d.

Slope of the curve

0.758

0.924

P(Chi²)

1.000

1.000

0.604

0.526

LOELR

500 mg/L

n.d.

NOELR

250 mg/L

n.d.

n.d.: not determined by statistical analysis

Endpoint(s) Effected:  Algal growth and biomass.

This information is used in a read-across approach in the assessment of the substance to be registered.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Description of key information

In a 72 h growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to the test substance at nominal concentrations of 0 (control), 125, 250, 500, 1000 and 2000 mg/L under static conditions in accordance with the OECD Guideline 201 (March 2006) and under GLP. Results based on growth rate after 72 h were as follows: Effect Loading rate (EL) 50: > 2000 mg/L; EL20: 464 mg/L (95 % C.l. 218 – 713 mg/L); EL10: 122 mg/L (95 % C.l. 19.0 – 247 mg/L); LOELR: 500 mg/L; NOELR: 250 mg/L. The values indicate effects at nominal loading rates. Due to methodological constraints, it was not possible to determine effect levels based on measured test item concentrations.

Measured concentrations were more than a factor of 1000 lower than in the respective nominal loading rates after pH adjustment. After pH adjustment the measured Ta5+ concentrations were low and showed high variability.

Concentrations of the test sample varied due to precipitation, agglomeration and adsorption reactions. Therefore, no concentration-response relationship can be delineated.

Observed growth inhibition effects may therefore rather predominantly refer to elevated NaCl concentrations, originated from neutralization, than released Ta5+ ions. Reported effects of sodium chloride concerning the freshwater alga P. subcapitata vary but inhibitory concentrations of 2.5 g/L, respectively 0.87 g/L have been determined (Geis et al., 2000 and Santos et al., 2007, in Heisterkamp, 2015). Due to a concentration of approximately 2.92 g/L NaCl, present at the highest loading rate of 2000 mg/L test substance, and the sensitivity of P. subcapitata to salinity, any hypothetical Ta5+ related effects cannot be distinguished from NaCl induced inhibition in the conducted test.

Key value for chemical safety assessment

Additional information

According to Annex VII, section 9.1.2, column 2 of the Regulation (EC) No 1907/2006, Growth inhibition studies on aquatic plants do not need to be conducted "if, there are mitigating factors indicating that aquatic toxicity is unlikely to occur, for instance if the substance is highly insoluble in water”. As the substance is expected to be highly insoluble (</= 21.3µg/L), see section 4.8 (Fox, 2012), the substance is not expected to be toxic to aquatic algae. Nevertheless, toxicity data from read-across sources are used to describe the toxicity.

This information is used in a read-across approach in the assessment of the substance to be registered.

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.